Combinations including CRY3AA and CRY6AA proteins to prevent development of resistance in corn rootworms (Diabrotica spp.)

ABSTRACT

The subject invention relates in part to Cry3Aa in combination with Cry6Aa. The subject invention relates in part to the surprising discovery that combinations of Cry3Aa and Cry6Aa are useful for preventing development of resistance (to either insecticidal protein system alone) by a corn rootworm ( Diabrotica  spp.) population. Included within the subject invention are plants producing these insecticidal Cry proteins, which are useful to mitigate concern that a corn rootworm population could develop that would be resistant to either of these insecticidal protein systems alone. The subject invention also relates in part to combinations of Cry3Aa and Cry6Aa proteins “triple-stacked” or “multi-stacked” with another insecticidal protein(s) such as a Cry6Aa protein or binary Cry34/35 proteins. Thus, such embodiments target rootworms with three modes of action. Transgenic plants, including corn, comprising a cry6Aa gene and a cry3Aa gene are included within the scope of the subject invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a national phase application, filed pursuant to 35 U.S.C. §371,of PCT application No. PCT/US2011/033622 filed on Apr. 22, 2011, whichclaims the benefit of U.S. provisional application No. 61/327,240, filedon Apr. 23, 2010, U.S. provisional application No. 61/388,273, filed onSep. 30, 2010, U.S. provisional application No. 61/476,005, filed onApr. 15, 2011, and U.S. provisional application No. 61/477,447, filed onApr. 20, 2011. The prior applications are incorporated herein byreference in their entirety.

BACKGROUND

Humans grow corn for food and energy applications. Corn is an importantcrop. It is an important source of food, food products, and animal feedin many areas of the world. Insects eat and damage plants and therebyundermine these human efforts. Billions of dollars are spent each yearto control insect pests and additional billions are lost to the damagethey inflict.

Damage caused by insect pests is a major factor in the loss of theworld's corn crops, despite the use of protective measures such aschemical pesticides. In view of this, insect resistance has beengenetically engineered into crops such as corn in order to controlinsect damage and to reduce the need for traditional chemicalpesticides.

Over 10 million acres of U.S. corn are infested with corn rootwormspecies complex each year. The corn rootworm species complex includesthe northern corn rootworm (Diabrotica barberi), the southern cornrootworm (D. undecimpunctata howardi), and the western corn rootworm (D.virgifera virgifera). (Other species include Diabrotica virgifera zeae(Mexican corn rootworm), Diabrotica balteata (Brazilian corn rootworm),and Brazilian corn rootworm complex (Diabrotica viridula and Diabroticaspeciosa).)

The soil-dwelling larvae of these Diabrotica species feed on the root ofthe corn plant, causing lodging. Lodging eventually reduces corn yieldand often results in death of the plant. By feeding on cornsilks, theadult beetles reduce pollination and, therefore, detrimentally affectthe yield of corn per plant. In addition, adults and larvae of the genusDiabrotica attack cucurbit crops (cucumbers, melons, squash, etc.) andmany vegetable and field crops in commercial production as well as thosebeing grown in home gardens.

Synthetic organic chemical insecticides have been the primary tools usedto control insect pests but biological insecticides, such as theinsecticidal proteins derived from Bacillus thuringiensis (Bt), haveplayed an important role in some areas. The ability to produceinsect-resistant plants through transformation with Bt insecticidalprotein genes has revolutionized modern agriculture and heightened theimportance and value of insecticidal proteins and their genes.

Insecticidal crystal proteins from some strains of Bacillusthuringiensis (B.t.) are well-known in the art. See, e.g., Hofte et al.,Microbial Reviews, Vol. 53, No. 2, pp. 242-255 (1989). These proteinsare typically produced by the bacteria as approximately 130 kDaprotoxins that are then cleaved by proteases in the insect midgut, afteringestion by the insect, to yield a roughly 60 kDa core toxin. Theseproteins are known as crystal proteins because distinct crystallineinclusions can be observed with spores in some strains of B.t. Thesecrystalline inclusions are often composed of several distinct proteins.

One group of genes which have been utilized for the production oftransgenic insect resistant crops are the delta-endotoxins from Bacillusthuringiensis (B.t.). Delta-endotoxins have been successfully expressedin crop plants such as cotton, potatoes, rice, sunflower, as well ascorn, and have proven to provide excellent control over insect pests.(Perlak, F. J et al. (1990) Bio/Technology 8, 939-943; Perlak, F. J. etal. (1993) Plant Mol. Biol. 22: 313-321; Fujimoto H. et al. (1993)Bio/Technology 11: 1151-1155; Tu et al. (2000) Nature Biotechnology18:1101-1104; PCT publication number WO 01/13731; and Bing J W et al.(2000) Efficacy of Cry1F Transgenic Maize, 14^(th) BiennialInternational Plant Resistance to Insects Workshop, Fort Collins, Colo.)

Several Bt proteins have been used to create the insect-resistanttransgenic plants that have been successfully registered andcommercialized to date. These include Cry1Ab, Cry1Ac, Cry1F, Cry1A.105,Cry2Ab, Cry3Aa, Cry3Bb, and Cry34/35Ab in corn, Cry1Ac and Cry2Ab incotton, and Cry3A in potato.

The commercial products expressing these proteins express a singleprotein except in cases where the combined insecticidal spectrum of 2proteins is desired (e.g., Cry1Ab and Cry3Bb in corn combined to provideresistance to lepidopteran pests and rootworm, respectively) or wherethe independent action of the proteins makes them useful as a tool fordelaying the development of resistance in susceptible insect populations(e.g., Cry1Ac and Cry2Ab in cotton combined to provide resistancemanagement for tobacco budworm).

Some of the qualities of insect-resistant transgenic plants that haveled to rapid and widespread adoption of this technology also give riseto the concern that pest populations will develop resistance to theinsecticidal proteins produced by these plants. Several strategies havebeen suggested for preserving the utility of Bt-based insect resistancetraits which include deploying proteins at a high dose in combinationwith a refuge, and alternation with, or co-deployment of, differenttoxins (McGaughey et al. (1998), “B.t. Resistance Management,” NatureBiotechnol. 16:144-146).

The proteins selected for use in an Insect Resistance Management (IRM)stack should be active such that resistance developed to one proteindoes not confer resistance to the second protein (i.e., there is notcross resistance to the proteins). If, for example, a pest populationselected for resistance to “Protein A” is sensitive to “Protein B”, onewould conclude that there is not cross resistance and that a combinationof Protein A and Protein B would be effective in delaying resistance toProtein A alone.

In the absence of resistant insect populations, assessments can be madebased on other characteristics presumed to be related tocross-resistance potential. The utility of receptor-mediated binding inidentifying insecticidal proteins likely to not exhibit cross resistancehas been suggested (van Mellaert et al. 1999). The key predictor of lackof cross resistance inherent in this approach is that the insecticidalproteins do not compete for receptors in a sensitive insect species.

In the event that two Bt toxins compete for the same receptor, then ifthat receptor mutates in that insect so that one of the toxins no longerbinds to that receptor and thus is no longer insecticidal against theinsect, it might be the case that the insect will also be resistant tothe second toxin (which competitively bound to the same receptor). Thatis, the insect is said to be cross-resistant to both Bt toxins. However,if two toxins bind to two different receptors, this could be anindication that the insect would not be simultaneously resistant tothose two toxins.

A relatively newer insecticidal protein system was discovered inBacillus thuringiensis as disclosed in WO 97/40162. This systemcomprises two proteins—one of approximately 14-15 kDa and the other ofabout 44-45 kDa. See also U.S. Pat. Nos. 6,083,499 and 6,127,180. Theseproteins have now been assigned to their own classes, and accordinglyreceived the Cry designations of Cry34 and Cry35, respectively. SeeCrickmore et al. website (biols.susx.ac.uk/home/Neil_Crickmore/Bt/).Many other related proteins of this type of system have now beendisclosed. See e.g. U.S. Pat. No. 6,372,480; WO 01/14417; and WO00/66742. Plant-optimized genes that encode such proteins, wherein thegenes are engineered to use codons for optimized expression in plants,have also been disclosed. See e.g. U.S. Pat. No. 6,218,188.

The exact mode of action of the Cry34/35 system has yet to bedetermined, but it appears to form pores in membranes of insect gutcells. See Moellenbeck et al., Nature Biotechnology, vol. 19, p. 668(July 2001); Masson et al., Biochemistry, 43 (12349-12357) (2004). Theexact mechanism of action remains unclear despite 3D atomic coordinatesand crystal structures being known for a Cry34 and a Cry35 protein. SeeU.S. Pat. Nos. 7,524,810 and 7,309,785. For example, it is unclear ifone or both of these proteins bind a typical type of receptor, such asan alkaline phosphatase or an aminopeptidase.

Furthermore, because there are different mechanisms by which an insectcan develop resistance to a Cry protein (such as by alteredglycosylation of the receptor [see Jurat-Fuentes et al. (2002) 68 AEM5711-5717], by removal of the receptor protein [see Lee et al. (1995) 61AEM 3836-3842], by mutating the receptor, or by other mechanisms [seeHeckel et al., J. Inv. Pathol. 95 (2007) 192-197]), it was impossible toa priori predict whether there would be cross-resistance betweenCry34/35 and other Cry proteins. Lefko et al. discusses a complexresistance phenomenon in rootworm. J. Appl. Entomol. 132 (2008) 189-204.

Predicting competitive binding for the Cry34/35 system is also furthercomplicated by the fact that two proteins are involved in the Cry34/35binary system. Again, it is unclear if and how these proteinseffectively bind the insect gut/gut cells, and if and how they interactwith or bind with each other.

Other options for controlling coleopterans include Cry3Bb toxins, Cry3C,Cry6B, ET29, ET33 with ET34, TIC407, TIC435, TIC417, TIC901, TIC1201,ET29 with TIC810, ET70, ET76 with ET80, TIC851, and others. RNAiapproaches have also been proposed. See e.g. Baum et al., NatureBiotechnology, vol. 25, no. 11 (November 2007) pp. 1322-1326.

Meihls et al. suggest the use of refuges for resistance management incorn rootworm. PNAS (2008) vol. 105, no. 49, 19177-19182.

BRIEF SUMMARY

The subject invention relates in part to Cry3Aa in combination withCry6Aa. The subject invention relates in part to the surprisingdiscovery that Cry3Aa and Cry6Aa are useful for preventing developmentof resistance (to either insecticidal protein system alone) by a cornrootworm (Diabrotica spp.) population. As one skilled in the art willrecognize with the benefit of this disclosure, plants producing theseinsecticidal Cry proteins will be useful to mitigate concern that a cornrootworm population could develop that would be resistant to either ofthese insecticidal protein systems alone.

The subject invention is supported in part by the discovery thatcomponents of these Cry protein systems do not compete with each otherfor binding corn rootworm gut receptors.

The subject invention also relates in part to triple stacks or“pyramids” of three (or more) toxin systems, with Cry3Aa and Cry6Aabeing the base pair. Thus, plants (and acreage planted with such plants)that produce these two insecticidal protein systems are included withinthe scope of the subject invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Binding of ¹²⁵I-Cry3Aa trypsin core (FIG. 1A) and ¹²⁵I-Cry6Aa1(full-length) (FIG. 1B) as a function of input radiolabeled Cry toxinsto BBMV prepared from western corn rootworm larvae. Specificbinding=total binding−non-specific binding, error bar=SEM (standarderror of mean).

FIG. 2. Binding of ¹²⁵I-Cry3Aa to BBMV prepared from western cornrootworm larvae at different concentrations of non-labeled competitor(log 0.1=−1.0, log 1=0, log 10=1.0, log 100=2.0, log 1000=3.0).

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1: Full-length Cry3Aa protein sequence

SEQ ID NO:2: Cry3Aa trypsin core protein sequence

SEQ ID NO:3: Full-length Cry6Aa1 protein sequence

DETAILED DESCRIPTION

Sequences for Cry3Aa and Cry6Aa proteins are obtainable from Bacillusthruingiensis isolates as listed on the Crickmore et al. website(lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/intro.html).

The subject invention includes the use of Cry3Aa insecticidal proteinsin combination with a Cry6Aa toxin to protect corn from damage and yieldloss caused by corn rootworm feeding by corn rootworm populations thatmight develop resistance to either of these Cry protein systems alone(without the other).

The subject invention thus teaches an Insect Resistance Management (IRM)stack to prevent the development of resistance by corn rootworm toCry6Aa and/or Cry3Aa.

The present invention provides compositions for controlling rootwormpests comprising cells that produce a Cry6Aa insecticidal protein and aCry3Aa insecticidal protein.

The invention further comprises a host transformed to produce both aCry6Aa protein and a Cry3Aa insecticidal protein, wherein said host is amicroorganism or a plant cell.

It is additionally intended that the invention provides a method ofcontrolling rootworm pests comprising contacting said pests or theenvironment of said pests with an effective amount of a composition thatcontains a Cry6Aa protein and further contains a Cry3Aa insecticidalprotein.

An embodiment of the invention comprises a maize plant comprising aplant-expressible gene encoding a Cry3Aa insecticidal protein and aplant-expressible gene encoding a Cry6Aa protein, and seed of such aplant.

A further embodiment of the invention comprises a maize plant wherein aplant-expressible gene encoding a Cry3Aa insecticidal protein and aplant-expressible gene encoding a Cry6Aa protein have been introgressedinto said maize plant, and seed of such a plant.

As described in the Examples, competitive receptor binding studies usingradiolabeled Cry3Aa core toxin protein show that the Cry6Aa core toxinprotein does not compete for binding in CRW insect tissue samples towhich Cry3Aa binds. See FIG. 2. These results indicate that thecombination of Cry6Aa and Cry3Aa proteins is an effective means tomitigate the development of resistance in CRW populations to eitherprotein system alone.

Thus, based in part on the data described above and elsewhere herein,Cry3Aa and Cry6Aa proteins can be used to produce IRM combinations forprevention or mitigation of resistance development by CRW. Otherproteins can be added to this combination to expand insect-controlspectrum, for example. The subject combination can also be used in somepreferred “triple stacks” or “pyramid” in combination with yet anotherprotein for controlling rootworms, such as Cry3Ba or binary Cry34/35proteins. Such additional combinations would thus provide multiple modesof action against a rootworm. RNAi against rootworms is a still furtheroption. See e.g. Baum et al., Nature Biotechnology, vol. 25, no. 11(November 2007) pp. 1322-1326.

In light of the disclosure of U.S. Ser. No. 61/327,240 (filed Apr. 23,2010) relating to combinations of Cry34Ab/35Ab and Cry3Aa proteins, U.S.Ser. No. 61/388,273 (filed Sep. 30, 2010) relating to combinations ofCry34Ab/35Ab and Cry6Aa proteins, and U.S. Ser. No. 61/476,005 (filedApr. 15, 2011) relating to combinations of Cry34Ab/35Ab and Cry3Baproteins, some preferred “triple stacks” or “multiple modes of actionstacks” of the subject invention include a Cry6Aa protein combined witha Cry3Aa protein, together with and a Cry3Ba protein or binary Cry34/35proteins. Transgenic plants, including corn, comprising a cry6Aa geneand a cry3Aa gene (optionally with a third or fourth toxin system, e.g.,Cry3B and/or Cry34/35) are included within the scope of the subjectinvention. Thus, such embodiments target the insect with at least threemodes of action.

Deployment options of the subject invention include the use of Cry6Aaand Cry3Aa proteins in corn-growing regions where Diabrotica spp. areproblematic. Another deployment option would be to use one or both ofthe Cry6Aa and Cry3Aa proteins in combination with other traits.

A person skilled in this art will appreciate that Bt toxins, even withina certain class such as Cry6Aa and Cry3Aa can vary to some extent.

Genes and Toxins.

The term “isolated” refers to a polynucleotide in a non-naturallyoccurring construct, or to a protein in a purified or otherwisenon-naturally occurring state. The genes and toxins useful according tothe subject invention include not only the full length sequencesdisclosed but also fragments of these sequences, variants, mutants, andfusion proteins which retain the characteristic pesticidal activity ofthe toxins specifically exemplified herein. As used herein, the terms“variants” or “variations” of genes refer to nucleotide sequences whichencode the same toxins or which encode equivalent toxins havingpesticidal activity. As used herein, the term “equivalent toxins” refersto toxins having the same or essentially the same biological activityagainst the target pests as the claimed toxins. The same applies toCry3B's and Cry34/35 if used in triple/multiple stacks according to thesubject invention. Domains/subdomains of these proteins can be swappedto make chimeric proteins, for example. See e.g. U.S. Pat. Nos.7,309,785 and 7,524,810 regarding Cry34/35 proteins. The '785 patentalso teaches truncated Cry35 proteins. Truncated toxins are alsoexemplified herein.

As used herein, the boundaries represent approximately 95% (Cry6Aa's andCry3Aa's), 78% (Cry6A's and Cry 3A's), and 45% (Cry6's and Cry 3's)sequence identity, per “Revision of the Nomenclature for the Bacillusthuringiensis Pesticidal Crystal Proteins,” N. Crickmore, D. R. Zeigler,J. Feitelson, E. Schnepf, J. Van Rie, D. Lereclus, J. Baum, and D. H.Dean. Microbiology and Molecular Biology Reviews (1998) Vol 62: 807-813.The same applies to Cry3B's and Cry34/35 (e.g. Cry34Ab/Cry35Ab) if usedin triple/multiple stacks, for example, according to the subjectinvention.

It should be apparent to a person skilled in this art that genesencoding active toxins can be identified and obtained through severalmeans. The specific genes or gene portions exemplified herein may beobtained from the isolates deposited at a culture depository. Thesegenes, or portions or variants thereof, may also be constructedsynthetically, for example, by use of a gene synthesizer. Variations ofgenes may be readily constructed using standard techniques for makingpoint mutations. Also, fragments of these genes can be made usingcommercially available exonucleases or endonucleases according tostandard procedures. For example, enzymes such as Bal31 or site-directedmutagenesis can be used to systematically cut off nucleotides from theends of these genes. Genes that encode active fragments may also beobtained using a variety of restriction enzymes. Proteases may be usedto directly obtain active fragments of these protein toxins.

Fragments and equivalents which retain the pesticidal activity of theexemplified toxins would be within the scope of the subject invention.Also, because of the redundancy of the genetic code, a variety ofdifferent DNA sequences can encode the amino acid sequences disclosedherein. It is well within the skill of a person trained in the art tocreate these alternative DNA sequences encoding the same, or essentiallythe same, toxins. These variant DNA sequences are within the scope ofthe subject invention. As used herein, reference to “essentially thesame” sequence refers to sequences which have amino acid substitutions,deletions, additions, or insertions which do not materially affectpesticidal activity. Fragments of genes encoding proteins that retainpesticidal activity are also included in this definition.

A further method for identifying the genes encoding the toxins and geneportions useful according to the subject invention is through the use ofoligonucleotide probes. These probes are detectable nucleotidesequences. These sequences may be detectable by virtue of an appropriatelabel or may be made inherently fluorescent as described inInternational Application No. WO93/16094. As is well known in the art,if the probe molecule and nucleic acid sample hybridize by forming astrong bond between the two molecules, it can be reasonably assumed thatthe probe and sample have substantial homology. Preferably,hybridization is conducted under stringent conditions by techniqueswell-known in the art, as described, for example, in Keller, G. H., M.M. Manak (1987) DNA Probes, Stockton Press, New York, N.Y., pp. 169-170.Some examples of salt concentrations and temperature combinations are asfollows (in order of increasing stringency): 2×SSPE or SSC at roomtemperature; 1×SSPE or SSC at 42° C.; 0.1×SSPE or SSC at 42° C.;0.1×SSPE or SSC at 65° C. Detection of the probe provides a means fordetermining in a known manner whether hybridization has occurred. Such aprobe analysis provides a rapid method for identifying toxin-encodinggenes of the subject invention. The nucleotide segments which are usedas probes according to the invention can be synthesized using a DNAsynthesizer and standard procedures. These nucleotide sequences can alsobe used as PCR primers to amplify genes of the subject invention.

Variant Toxins.

Certain toxins of the subject invention have been specificallyexemplified herein. Since these toxins are merely exemplary of thetoxins of the subject invention, it should be readily apparent that thesubject invention comprises variant or equivalent toxins (and nucleotidesequences coding for equivalent toxins) having the same or similarpesticidal activity of the exemplified toxin. Equivalent toxins willhave amino acid homology with an exemplified toxin. This amino acididentity will typically be greater than 75%, or preferably greater than85%, preferably greater than 90%, preferably greater than 95%,preferably greater than 96%, preferably greater than 97%, preferablygreater than 98%, or preferably greater than 99% in some embodiments.The amino acid identity will typically be highest in critical regions ofthe toxin which account for biological activity or are involved in thedetermination of three-dimensional configuration which ultimately isresponsible for the biological activity. In this regard, certain aminoacid substitutions are acceptable and can be expected if thesesubstitutions are in regions which are not critical to activity or areconservative amino acid substitutions which do not affect thethree-dimensional configuration of the molecule. For example, aminoacids may be placed in the following classes: non-polar, unchargedpolar, basic, and acidic. Conservative substitutions whereby an aminoacid of one class is replaced with another amino acid of the same typefall within the scope of the subject invention so long as thesubstitution does not materially alter the biological activity of thecompound. Table 1 provides a listing of examples of amino acidsbelonging to each class.

TABLE 1 Class of Amino Acid Examples of Amino Acids Nonpolar Ala, Val,Leu, Ile, Pro, Met, Phe, Trp Uncharged Polar Gly, Ser, Thr, Cys, Tyr,Asn, Gln Acidic Asp, Glu Basic Lys, Arg, His

In some instances, non-conservative substitutions can also be made. Thecritical factor is that these substitutions must not significantlydetract from the biological activity of the toxin.

Recombinant Hosts.

The genes encoding the toxins of the subject invention can be introducedinto a wide variety of microbial or plant hosts. Expression of the toxingene results, directly or indirectly, in the intracellular productionand maintenance of the pesticide. Conjugal transfer and recombinanttransfer can be used to create a Bt strain that expresses both toxins ofthe subject invention. Other host organisms may also be transformed withone or both of the toxin genes then used to accomplish the synergisticeffect. With suitable microbial hosts, e.g., Pseudomonas, the microbescan be applied to the situs of the pest, where they will proliferate andbe ingested. The result is control of the pest. Alternatively, themicrobe hosting the toxin gene can be treated under conditions thatprolong the activity of the toxin and stabilize the cell. The treatedcell, which retains the toxic activity, then can be applied to theenvironment of the target pest. Non-regenerable/non-totipotent plantcells from a plant of the subject invention (comprising at least one ofthe subject IRM genes) are included within the subject invention.

Plant Transformation.

A preferred embodiment of the subject invention is the transformation ofplants with genes encoding the subject insecticidal protein or itsvariants. The transformed plants are resistant to attack by an insecttarget pest by virtue of the presence of controlling amounts of thesubject insecticidal protein or its variants in the cells of thetransformed plant. By incorporating genetic material that encodes theinsecticidal properties of the B.t. insecticidal toxins into the genomeof a plant eaten by a particular insect pest, the adult or larvae woulddie after consuming the food plant. Numerous members of themonocotyledonous and dicotyledonous classifications have beentransformed. Transgenic agronomic crops as well as fruits and vegetablesare of commercial interest. Such crops include, but are not limited to,maize, rice, soybeans, canola, sunflower, alfalfa, sorghum, wheat,cotton, peanuts, tomatoes, potatoes, and the like. Several techniquesexist for introducing foreign genetic material into plant cells, and forobtaining plants that stably maintain and express the introduced gene.Such techniques include acceleration of genetic material coated ontomicroparticles directly into cells (U.S. Pat. No. 4,945,050 and U.S.Pat. No. 5,141,131). Plants may be transformed using Agrobacteriumtechnology, see U.S. Pat. No. 5,177,010, U.S. Pat. No. 5,104,310,European Patent Application No. 0131624B1, European Patent ApplicationNo. 120516, European Patent Application No. 159418B1, European PatentApplication No. 176112, U.S. Pat. No. 5,149,645, U.S. Pat. No.5,469,976, U.S. Pat. No. 5,464,763, U.S. Pat. No. 4,940,838, U.S. Pat.No. 4,693,976, European Patent Application No. 116718, European PatentApplication No. 290799, European Patent Application No. 320500, EuropeanPatent Application No. 604662, European Patent Application No. 627752,European Patent Application No. 0267159, European Patent Application No.0292435, U.S. Pat. No. 5,231,019, U.S. Pat. No. 5,463,174, U.S. Pat. No.4,762,785, U.S. Pat. No. 5,004,863, and U.S. Pat. No. 5,159,135. Othertransformation technology includes WHISKERS™ technology, see U.S. Pat.No. 5,302,523 and U.S. Pat. No. 5,464,765. Electroporation technologyhas also been used to transform plants, see WO 87/06614, U.S. Pat. No.5,472,869, U.S. Pat. No. 5,384,253, WO 9209696, and WO 9321335. All ofthese transformation patents and publications are incorporated byreference. In addition to numerous technologies for transforming plants,the type of tissue which is contacted with the foreign genes may vary aswell. Such tissue would include but would not be limited to embryogenictissue, callus tissue types I and II, hypocotyl, meristem, and the like.Almost all plant tissues may be transformed during dedifferentiationusing appropriate techniques within the skill of an artisan.

Genes encoding any of the subject toxins can be inserted into plantcells using a variety of techniques which are well known in the art asdisclosed above. For example, a large number of cloning vectorscomprising a marker that permits selection of the transformed microbialcells and a replication system functional in Escherichia coli areavailable for preparation and modification of foreign genes forinsertion into higher plants. Such manipulations may include, forexample, the insertion of mutations, truncations, additions, orsubstitutions as desired for the intended use. The vectors comprise, forexample, pBR322, pUC series, M13 mp series, pACYC184, etc. Accordingly,the sequence encoding the Cry protein or variants can be inserted intothe vector at a suitable restriction site. The resulting plasmid is usedfor transformation of cells of E. coli, the cells of which arecultivated in a suitable nutrient medium, then harvested and lysed sothat workable quantities of the plasmid are recovered. Sequenceanalysis, restriction fragment analysis, electrophoresis, and otherbiochemical-molecular biological methods are generally carried out asmethods of analysis. After each manipulation, the DNA sequence used canbe cleaved and joined to the next DNA sequence. Each manipulated DNAsequence can be cloned in the same or other plasmids.

The use of T-DNA-containing vectors for the transformation of plantcells has been intensively researched and sufficiently described in EP120516; Lee and Gelvin (2008), Fraley et al. (1986), and An et al.(1985), and is well established in the field.

Once the inserted DNA has been integrated into the plant genome, it isrelatively stable throughout subsequent generations. The vector used totransform the plant cell normally contains a selectable marker geneencoding a protein that confers on the transformed plant cellsresistance to a herbicide or an antibiotic, such as bialaphos,kanamycin, G418, bleomycin, or hygromycin, inter alia. The individuallyemployed selectable marker gene should accordingly permit the selectionof transformed cells while the growth of cells that do not contain theinserted DNA is suppressed by the selective compound.

A large number of techniques are available for inserting DNA into a hostplant cell. Those techniques include transformation with T-DNA deliveredby Agrobacterium tumefaciens or Agrobacterium rhizogenes as thetransformation agent. Additionally, fusion of plant protoplasts withliposomes containing the DNA to be delivered, direct injection of theDNA, biolistics transformation (microparticle bombardment), orelectroporation, as well as other possible methods, may be employed.

In a preferred embodiment of the subject invention, plants will betransformed with genes wherein the codon usage of the protein codingregion has been optimized for plants. See, for example, U.S. Pat. No.5,380,831, which is hereby incorporated by reference. Also,advantageously, plants encoding a truncated toxin will be used. Thetruncated toxin typically will encode about 55% to about 80% of the fulllength toxin. Methods for creating synthetic B.t. genes for use inplants are known in the art (Stewart, 2007).

Regardless of transformation technique, the gene is preferablyincorporated into a gene transfer vector adapted to express the B.tinsecticidal toxin genes and variants in the plant cell by including inthe vector a plant promoter. In addition to plant promoters, promotersfrom a variety of sources can be used efficiently in plant cells toexpress foreign genes. For example, one may use promoters of bacterialorigin, such as the octopine synthase promoter, the nopaline synthasepromoter, and the mannopine synthase promoter.Non-Bacillus-thuringiensis promoters can be used in some preferredembodiments. Promoters of plant virus origin may be used, for example,the ³⁵S and 19S promoters of Cauliflower Mosaic Virus, a promoter fromCassava Vein Mosaic Virus, and the like. Plant promoters include, butare not limited to, ribulose-1,6-bisphosphate (RUBP) carboxylase smallsubunit (ssu), beta-conglycinin promoter, phaseolin promoter, ADH(alcohol dehydrogenase) promoter, heat-shock promoters, ADF (actindepolymerization factor) promoter, ubiquitin promoter, actin promoter,and tissue specific promoters. Promoters may also contain certainenhancer sequence elements that may improve the transcriptionefficiency. Typical enhancers include but are not limited to ADH1-intron1 and ADH1-intron 6. Constitutive promoters may be used. Constitutivepromoters direct continuous gene expression in nearly all cells typesand at nearly all times (e.g., actin, ubiquitin, CaMV 35S). Tissuespecific promoters are responsible for gene expression in specific cellor tissue types, such as the leaves or seeds (e.g. zein, oleosin, napin,ACP (Acyl Carrier Protein) promoters), and these promoters may also beused. Promoters may also be used that are active during a certain stageof the plants' development as well as active in specific plant tissuesand organs. Examples of such promoters include but are not limited topromoters that are root specific, pollen-specific, embryo specific, cornsilk specific, cotton fiber specific, seed endosperm specific, phloemspecific, and the like.

Under certain circumstances it may be desirable to use an induciblepromoter. An inducible promoter is responsible for expression of genesin response to a specific signal, such as: physical stimulus (e.g. heatshock genes); light (e.g. RUBP carboxylase); hormone (e.g.glucocorticoid); antibiotic (e.g. tetracycline); metabolites; and stress(e.g. drought). Other desirable transcription and translation elementsthat function in plants may be used, such as 5′ untranslated leadersequences, RNA transcription termination sequences and poly-adenylateaddition signal sequences. Numerous plant-specific gene transfer vectorsare known to the art.

Transgenic crops containing insect resistance (IR) traits are prevalentin corn and cotton plants throughout North America, and usage of thesetraits is expanding globally. Commercial transgenic crops combining IRand herbicide tolerance (HT) traits have been developed by multiple seedcompanies. These include combinations of IR traits conferred by B.t.insecticidal proteins and HT traits such as tolerance to AcetolactateSynthase (ALS) inhibitors such as sulfonylureas, imidazolinones,triazolopyrimidine, sulfonanilides, and the like, Glutamine Synthetase(GS) inhibitors such as bialaphos, glufosinate, and the like,4-HydroxyPhenylPyruvate Dioxygenase (HPPD) inhibitors such asmesotrione, isoxaflutole, and the like,5-EnolPyruvylShikimate-3-Phosphate Synthase (EPSPS) inhibitors such asglyphosate and the like, and Acetyl-Coenzyme A Carboxylase (ACCase)inhibitors such as haloxyfop, quizalofop, diclofop, and the like. Otherexamples are known in which transgenically provided proteins provideplant tolerance to herbicide chemical classes such as phenoxy acidsherbicides and pyridyloxyacetates auxin herbicides (see WO 2007/053482A2), or phenoxy acids herbicides and aryloxyphenoxypropionatesherbicides (see WO 2005107437 A2, A3). The ability to control multiplepest problems through IR traits is a valuable commercial productconcept, and the convenience of this product concept is enhanced ifinsect control traits and weed control traits are combined in the sameplant. Further, improved value may be obtained via single plantcombinations of IR traits conferred by a B.t. insecticidal protein suchas that of the subject invention, with one or more additional HT traitssuch as those mentioned above, plus one or more additional input traits(e.g. other insect resistance conferred by B.t.-derived or otherinsecticidal proteins, insect resistance conferred by mechanisms such asRNAi and the like, nematode resistance, disease resistance, stresstolerance, improved nitrogen utilization, and the like), or outputtraits (e.g. high oils content, healthy oil composition, nutritionalimprovement, and the like). Such combinations may be obtained eitherthrough conventional breeding (breeding stack) or jointly as a noveltransformation event involving the simultaneous introduction of multiplegenes (molecular stack). Benefits include the ability to manage insectpests and improved weed control in a crop plant that provides secondarybenefits to the producer and/or the consumer. Thus, the subjectinvention can be used in combination with other traits to provide acomplete agronomic package of improved crop quality with the ability toflexibly and cost effectively control any number of agronomic issues.

The transformed cells grow inside the plants in the usual manner. Theycan form germ cells and transmit the transformed trait(s) to progenyplants.

Such plants can be grown in the normal manner and crossed with plantsthat have the same transformed hereditary factors or other hereditaryfactors. The resulting hybrid individuals have the correspondingphenotypic properties.

In a preferred embodiment of the subject invention, plants will betransformed with genes wherein the codon usage has been optimized forplants. See, for example, U.S. Pat. No. 5,380,831. In addition, methodsfor creating synthetic Bt genes for use in plants are known in the art(Stewart and Burgin, 2007). One non-limiting example of a preferredtransformed plant is a fertile maize plant comprising a plantexpressible gene encoding a Cry6Aa protein, and further comprising asecond set of plant expressible genes encoding Cry3Aa proteins.

Transfer (or introgression) of the Cry6Aa- and Cry3Aa-determinedtrait(s) into inbred maize lines can be achieved by recurrent selectionbreeding, for example by backcrossing. In this case, a desired recurrentparent is first crossed to a donor inbred (the non-recurrent parent)that carries the appropriate gene(s) for the Cry-determined traits. Theprogeny of this cross is then mated back to the recurrent parentfollowed by selection in the resultant progeny for the desired trait(s)to be transferred from the non-recurrent parent. After three, preferablyfour, more preferably five or more generations of backcrosses with therecurrent parent with selection for the desired trait(s), the progenywill be heterozygous for loci controlling the trait(s) beingtransferred, but will be like the recurrent parent for most or almostall other genes (see, for example, Poehlman & Sleper (1995) BreedingField Crops, 4th Ed., 172-175; Fehr (1987) Principles of CultivarDevelopment, Vol. 1: Theory and Technique, 360-376).

Insect Resistance Management (IRM) Strategies.

Roush et al., for example, outlines two-toxin strategies, also called“pyramiding” or “stacking,” for management of insecticidal transgeniccrops. (The Royal Society. Phil. Trans. R. Soc. Lond. B. (1998) 353,1777-1786).

On their website, the United States Environmental Protection Agency(epa.gov/oppbppd1/biopesticides/pips/bt_corn_refuge_2006.htm) publishesthe following requirements for providing non-transgenic (i.e., non-B.t.)refuges (a block of non-Bt crops/corn) for use with transgenic cropsproducing a single Bt protein active against target pests.

-   -   “The specific structured requirements for corn borer-protected        Bt (Cry1Ab or Cry1F) corn products are as follows:    -   Structured refuges:        -   20% non-Lepidopteran Bt corn refuge in Corn Belt;        -   50% non-Lepidopteran Bt refuge in Cotton Belt    -   Blocks        -   Internal (i.e., within the Bt field)        -   External (i.e., separate fields within ½ mile (¼ mile if            possible) of the Bt field to maximize random mating)    -   In-field Strips        -   Strips must be at least 4 rows wide (preferably 6 rows) to            reduce the effects of larval movement”

In addition, the National Corn Growers Association, on their website:

-   -   (ncga.com/insect-resistance-management-fact-sheet-bt-corn)        also provides similar guidance regarding the refuge        requirements. For example:    -   “Requirements of the Corn Borer IRM:    -   Plant at least 20% of your corn acres to refuge hybrids    -   In cotton producing regions, refuge must be 50%    -   Must be planted within ½ mile of the refuge hybrids    -   Refuge can be planted as strips within the Bt field; the refuge        strips must be at least 4 rows wide    -   Refuge may be treated with conventional pesticides only if        economic thresholds are reached for target insect    -   Bt-based sprayable insecticides cannot be used on the refuge        corn    -   Appropriate refuge must be planted on every farm with Bt corn”

As stated by Roush et al. (on pages 1780 and 1784 right column, forexample), stacking or pyramiding of two different proteins eacheffective against the target pests and with little or nocross-resistance can allow for use of a smaller refuge. Roush suggeststhat for a successful stack, a refuge size of less than 10% refuge, canprovide comparable resistance management to about 50% refuge for asingle (non-pyramided) trait. For currently available pyramided Bt cornproducts, the U.S. Environmental Protection Agency requiressignificantly less (generally 5%) structured refuge of non-Bt corn beplanted than for single trait products (generally 20%).

There are various ways of providing the IRM effects of a refuge,including various geometric planting patterns in the fields (asmentioned above) and in-bag seed mixtures, as discussed further by Roushet al. (supra), and U.S. Pat. No. 6,551,962.

The above percentages, or similar refuge ratios, can be used for thesubject double or triple stacks or pyramids.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety to the extent they are not inconsistent with theexplicit teachings of this specification.

Following are examples that illustrate procedures for practicing theinvention. These examples should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted. All temperatures are in degrees Celsius.

Unless specifically indicated or implied, the terms “a”, “an”, and “the”signify “at least one” as used herein.

EXAMPLES Example 1—Construction of Expression Plasmids Encoding Cry3Aaand Cry6Aa Full-Length Toxins

Standard cloning methods were used in the construction of Pseudomonasfluorescens (Pf) expression plasmids engineered to produce full-lengthCry3Aa and Cry6Aa Cry proteins, respectively. Restriction endonucleasesfrom New England BioLabs (NEB; Ipswich, Mass.) were used for DNAdigestion and T4 DNA Ligase from Invitrogen was used for DNA ligation.Plasmid preparations were performed using the Plasmid Mini kit (Qiagen,Valencia, Calif.), following the instructions of the supplier. DNAfragments were purified using the Millipore Ultrafree®-DA cartridge(Billerica, Mass.) after agarose Tris-acetate gel electrophoresis. Thebasic cloning strategy entailed subcloning the coding sequences (CDS) offull-length Cry proteins into pMYC1803 for Cry3Aa and into pDOW1169 forCry6Aa at SpeI and XhoI (or SalI that is compatible with XhoI)restriction sites respectively, whereby they were placed under theexpression control of the Ptac promoter and the rrnBT1T2 or rrnBT2terminator from plasmid pKK223-3 (PL Pharmacia, Milwaukee, Wis.)respectively. pMYC1803 is a medium copy number plasmid with the RSF1010origin of replication, a tetracycline resistance gene, and a ribosomebinding site preceding the restriction enzyme recognition sites intowhich DNA fragments containing protein coding regions may be introduced(US Patent Application No. 20080193974). The expression plasmid forCry3Aa (recombinant pMYC1803) was transformed by electroporation into aP. fluorescens strain MB214, recovered in SOC-Soy hydrolysate medium,and plated on Lysogeny broth (LB) medium containing 20 μg/mltetracycline. The expression vector pDOW1169 is similar to pMYC1803 butpDOW1169 carries the pyrF gene encoding uracil, which was used as amarker for screening for transformants when a P. fluorescens uracilauxotrophic strain (such as DPf10) was used for transformation on aplate of M9 minimal medium that lacked of uracil (Schneider et al.2005). Details of the microbiological manipulations are available fromUS Patent Application No. 20060008877, US Patent Application No.20080193974, and US Patent Application No. 20080058262, incorporatedherein by reference. Colonies were further screened by restrictiondigestion of miniprep plasmid DNA. Plasmid DNA of selected clonescontaining inserts was sequenced by contract with a commercialsequencing vendor such as eurofins MWG Operon (Huntsville, Ala.).Sequence data were assembled and analyzed using the Sequencher™ software(Gene Codes Corp., Ann Arbor, Mich.).

Example 2—Growth and Expression

Growth and expression analysis in shake-flask production of Cry3Aa andCry6Aa toxins for characterization including Bt receptor binding andinsect bioassay were accomplished by shake-flask-grown P. fluorescensstrains harboring expression constructs (e.g. clone pMYC1334 for Cry3Aaand pDAB102018 for Cry6Aa). Seed culture for Cry3Aa grown in P.fluorescens medium overnight supplemented with 20 μg/ml tetracycline wasused to inoculate 200 mL of the same medium with 20 μg/ml tetracycline.However, the seed culture for Cry6Aa was grown in M9 minimal brothovernight and was used to inoculate 200 mL of the P. fluorescens mediumwithout antibiotic. Expressions of Cry3Aa and Cry6Aa toxins via the Ptacpromoter were induced by addition ofisopropyl-β-D-1-thiogalactopyranoside (IPTG) after an initial incubationof 24 hours at 28-30° C. with shaking at 300 rpm. Cultures were sampledat the time of induction and at various times post-induction. Celldensity was measured by optical density at 600 nm (OD₆₀₀).

Example 3—Cell Fractionation and SDS-PAGE Analysis of Shake FlaskSamples

At each sampling time, the cell density of the samples was adjusted toOD₆₀₀=20 and 1-mL aliquots were centrifuged at 14,000×g for fiveminutes. The cell pellets were frozen at −80° C. Soluble and insolublefractions from frozen cell pellet samples were generated using EasyLyse™Bacterial Protein Extraction Solution (EPICENTRE® Biotechnologies,Madison, Wis.). Each cell pellet was resuspended in 1 mL EasyLyse™solution and further diluted 1:4 in lysis buffer and incubated withshaking at room temperature for 30 minutes. The lysate was centrifugedat 14,000 rpm for 20 minutes at 4° C. and the supernatant was recoveredas the soluble fraction. The pellet (insoluble fraction) was thenresuspended in an equal volume of phosphate buffered saline (PBS; 11.9mM Na₂HPO₄, 137 mM NaCl, 2.7 mM KCl, pH7.4). Samples were mixed at 3:1with 4× Laemmli sample buffer containing β-mercaptoethanol and boiledfor 5 minutes prior to loading onto NuPAGE Novex 4-20% Bis-Tris gels(Invitrogen, Carlsbad, Calif.). Electrophoresis was performed in therecommended NuPAGE MOPS buffer. Gels were stained with the SimplyBlue™Safe Stain according to the manufacturer's (Invitrogen) protocol andimaged using the Typhoon imaging system (GE Healthcare Life Sciences,Pittsburgh, Pa.).

Example 4—Inclusion Body Preparation

Cry protein inclusion body (IB) preparations were performed from P.fluorescens fermentations that produced insoluble B.t. insecticidalprotein, as demonstrated by SDS-PAGE and MALDI-MS (Matrix Assisted LaserDesorption/Ionization Mass Spectrometry). P. fluorescens cell pelletscreated from 48 hours post induction were thawed in a 37° C. water bath.The cells were resuspended to 25% w/v in lysis buffer (50 mM Tris, pH7.5, 200 mM NaCl, 20 mM EDTA disodium salt (Ethylenediaminetetraaceticacid), 1% Triton X-100, and 5 mM Dithiothreitol (DTT); 5 mL/L ofbacterial protease inhibitor cocktail (P8465 Sigma-Aldrich, St. Louis,Mo.) was added just prior to use only for Cry3Aa. The cells weresuspended using a homogenizer at lowest setting (Tissue Tearor, BioSpecProducts, Inc., Bartlesville, Okla.). Twenty five mg of lysozyme (SigmaL7651, from chicken egg white) was added to the cell suspension bymixing with a metal spatula, and the suspension was incubated at roomtemperature for one hour. The suspension was cooled on ice for 15minutes, then sonicated using a Branson Sonifier 250 (two 1-minutesessions, at 50% duty cycle, 30% output). Cell lysis was checked bymicroscopy. An additional 25 mg of lysozyme was added if necessary, andthe incubation and sonication were repeated. When cell lysis wasconfirmed via microscopy, the lysate was centrifuged at 11,500×g for 25minutes (4° C.) to form the IB pellet, and the supernatant wasdiscarded. The IB pellet was resuspended with 100 mL lysis buffer,homogenized with the hand-held mixer and centrifuged as above. The IBpellet was repeatedly washed by resuspension (in 50 mL lysis buffer),homogenization, sonication, and centrifugation until the supernatantbecame colorless and the IB pellet became firm and off-white in color.For the final wash, the IB pellet was resuspended in sterile-filtered(0.22 μm) distilled water containing 2 mM EDTA, and centrifuged. Thefinal pellet was resuspended in sterile-filtered distilled watercontaining 2 mM EDTA, and stored in 1 mL aliquots at −80° C.

Example 5—SDS-PAGE Analysis and Quantification

SDS-PAGE analysis and quantification of protein in IB preparations weredone by thawing a 1 mL aliquot of IB pellet and diluting 1:20 withsterile-filtered distilled water. The diluted sample was then boiledwith 4× reducing sample buffer [250 mM Tris, pH6.8, 40% glycerol (v/v),0.4% Bromophenol Blue (w/v), 8% SDS (w/v) and 8% β-Mercapto-ethanol(v/v)] and loaded onto a Novex® 4-20% Tris-Glycine, 12+2 well gel(Invitrogen) run with 1× Tris/Glycine/SDS buffer (Invitrogen). The gelwas run for approximately 60 min at 200 volts then stained and destainedby following the SimplyBlue™ Safe Stain (Invitrogen) procedures.Quantification of target bands was done by comparing densitometricvalues for the bands against Bovine Serum Albumin (BSA) samples run onthe same gel to generate a standard curve using the Bio-Rad Quantity Onesoftware.

Example 6—Solubilization of Inclusion Bodies

Ten mL of inclusion body suspensions from P. fluorescens clones MR832and DPf13032 (containing 50-70 mg/mL of Cry3Aa and Cry6Aa proteinsrespectively) were centrifuged at the highest setting of an Eppendorfmodel 5415C microfuge (approximately 14,000×g) to pellet the inclusions.The storage buffer supernatant was removed and replaced with 25 mL 50 mMCAPS [3-(cyclohexamino)1-propanesulfonic acid] buffer, pH10.5, for bothCry3Aa and Cry6Aa, in a 50 mL conical tube respectively. Inclusions wereresuspended using a pipette and vortexed to mix thoroughly. The tubeswere placed on a gently rocking platform at 4° C. overnight to extractfull-length Cry3Aa and Cry6Aa proteins. The extracts were centrifuged at30,000×g for 30 min at 4° C., and saved the resulting supernatantscontaining solubilized full-length Cry proteins.

Example 7—Truncation of Full-Length Protoxin

Full-length Cry3Aa was digested with trypsin to generate an active formof the Cry protein resistant to further trypsin digestion. Specifically,the solubilized full-length Cry3Aa was incubated with trypsin (bovinepancreas) (Sigma, St. MO) at (20:1=Cry protein:enzyme, w/w) in the 100mM sodium carbonate buffer, pH11, at room temperature for 1-3 hours.Complete activation or truncation was confirmed by SDS-PAGE analysis.The molecular mass of the full-length Cry3Aa was ≈73 kDa, and thetrypsin core was ≈55 kDa, respectively. The amino acid sequences of thefull-length and trypsin core of Cry3Aa are provided as SEQ ID 1 and SEQID 2. The full-length Cry6Aa is significantly more active to targetinsect corn rootworm than its either chymotrypsin or trypsin core. Thus,the full-length Cry6Aa was used for binding assays. The amino acidsequence of the full-length Cry6Aa is provided as SEQ ID 3.

Example 8—Purification of Cry Toxins

The trypsinized Cry3Aa and full-length Cry6Aa were further purifiedusing an ion-exchange chromatography system. Specifically, they werefurther purified using ATKA Explorer liquid chromatography system(Amersham Biosciences). For both Cry3Aa and Cry6Aa, buffer A was 10 mMCAPS buffer, pH 10.5, and buffer B was 10 mM CAPS buffer, pH 10.5+1 MNaCl. A Capto Q (5 ml) column (GE) was used. After the column was fullyequilibrated using the buffer A, a Cry toxin solution was injected intothe column at a flow rate of 5 ml/min. Elution was performed usinggradient 0-100% of the buffer B at 5 ml/min with 1 ml/fraction. AfterSDS-PAGE analysis of the selected fractions to further select fractionscontaining the best quality target protein, pooled those fractions. Thebuffer was changed for the both purified Cry3Aa trypsin core andfull-length Cry6Aa to 10 mM CAPS, pH 10.5 through dialysis. The sampleswere saved at 4° C. for later binding assay after being quantified usingSDS-PAGE and the Typhoon imaging system (GE) analyses with BSA as astandard.

Example 9—BBMV Preparations

Brush border membrane vesicle (BBMV) preparations of insect midguts havebeen widely used for Cry toxin receptor binding assays. The BBMVpreparations used in this invention were prepared from isolated midgutsof third instars of the western corn rootworm (Diabrotica virgiferavirgifera LeConte) using the method described by Wolfersberger et al.(1987). Leucine aminopeptidase was used as a marker of membrane proteinsin the preparation and Leucine aminopeptidase activities of crudehomogenate and BBMV preparation were determined as previously described(Li et al. 2004a). Protein concentration of the BBMV preparation wasmeasured using the Bradford method (1976).

Example 10—125I Labeling

Purified Cry3Aa trypsin core and full-length Cry6Aa were labeled using¹²⁵I for saturation and competition binding assays. To ensure theradio-labeling does not abolish the biological activity of the Crytoxins, cold iodination was conducted using NaI by following theinstructions of Pierce® Iodination Beads (Pierce Biotechnology, ThermoScientific, Rockford Ill.). Bioassay results indicated that bothiodinated Cry3Aa trypsin core and full-length Cry6Aa remained activeagainst the larvae of the western corn rootworm. ¹²⁵I-Cry3Aa and¹²⁵I-Cry6Aa were obtained with Pierce® Iodination Beads (Pierce) andNa¹²⁵I. Zeba™ Desalt Spin Columns (Pierce) were used to removeunincorporated or free Na¹²⁵I from the iodinated protein. The specificradio-activities of the iodinated Cry proteins ranged from 1-5 uCi/ug.Multiple replicates of labeling and binding assays were conducted.

Example 11—Saturation Binding Assays

Saturation binding assays were performed using ¹²⁵I-labeled Cry toxinsas described previously (Li et al. 2004b). To determine specific bindingand estimate the binding affinity (disassociation constant, Kd) andbinding site concentration (the maximum amount of toxin specificallybound to a given amount of BBMV, Bmax) of Cry3Aa and Cry6Aa to theinsect BBMV, a series of increasing concentrations of either ¹²⁵I-Cry3Aaor ¹²⁵I-Cry6Aa were incubated with a given concentration (0.1 mg/ml) ofthe insect BBMV respectively, in 150 ul of 20 mM Bis-Tris, pH 6.0, 150mM KCl, supplemented with 0.1% BSA at room temperature for 60 min withgentle shaking Toxin bound to BBMV was separated from free toxins in thesuspension by centrifugation at 20,000×g at room temperature for 8 min.The pellet was washed twice with 900 ul of ice-cold the same buffercontaining 0.1% BSA. The radio-activity remaining in the pellet wasmeasured with a COBRAII Auto-Gamma counter (Packard, a Can berracompany) and considered total binding. Another series of bindingreactions were setup at side by side, and a 500-1,000-fold excess ofunlabeled corresponding toxin was included in each of the bindingreactions to fully occupy all specific binding sites on the BBMV, whichwas used to determine non-specific binding. Specific binding wasestimated by subtracting the non-specific binding from the totalbinding. The Kd and Bmax values of these toxins were estimated using thetoxin molecule number (pmole) specifically bound to per microgram BBMVprotein against the concentrations of the labeled toxin used by runningGraphPad Prism 5.01 (GraphPad Software, San Diego, Calif.). The chartswere made using either Microsoft Excel or GraphPad Prism program. Theexperiments were replicated at least three times. These bindingexperiments demonstrated that both ¹²⁵I-Cry3Aa and ¹²⁵I-Cry6Aa were ableto specifically bind to the BBMV (FIGS. 1A and 1B). ¹²⁵I-Cry3Aa and¹²⁵I-Cry6Aa had a binding affinity Kd=24.0±9.76, 10.14±8.29 (nM), and abinding site concentration Bmax=21.97±5.99 pmole/mg, 0.66±0.33 (pmole/mgBBMV), respectively.

Example 12—Competition Binding Assays

Competition binding assays were further conducted to determine if Cry3Aatrypsin core and full-length Cry6Aa share BBMV binding sites. For Cry3Aahomologous competition binding assays, increasing amounts (0-2500 nM) ofunlabeled Cry3Aa were first mixed with 5 nM labeled Cry3Aa, and thenincubated with a given concentration (0.1 mg/ml) of BBMV at roomtemperature for 60 min, respectively. The percentages of bound¹²⁵I-Cry3Aa with BBMV were determined for each of the reactions ascompared to the initial specific binding at absence of unlabeledcompetitor. Heterologous competition binding assay between ¹²⁵I-Cry3Aaand unlabeled Cry6Aa was performed to identify if they share a same setof receptor(s). This was achieved by increasing the amount of unlabeledCry6Aa as a competitor included in the reactions to compete for theputative receptor(s) on the BBMV with the labeled Cry3Aa. The experimentwas replicated at least three times. The experimental resultsdemonstrated that Cry3Aa was able to displace itself about 60% when themolar concentration increased to approximately 2500 nM (500 folds excesscompared to 5 nM ¹²⁵I-Cry3Aa). The remaining about 40% was considerednonspecific binding that was not able to be displaced based on thesaturation binding result described elsewhere and the definition ofnon-specific binding, and thus the non-specific binding was subtractedand not shown here. This suggests that the specific binding wascompletely displaced by 500-fold excess unlabeled Cry3Aa (FIG. 2).However, Cry6Aa was not able to displace ¹²⁵I-Cry3Aa. These dataindicate that Cry3Aa does not share a receptor or a binding site withCry6Aa.

REFERENCES

-   Bradford, M. M. 1976. A rapid and sensitive method for the    quantitation of microgram quantities of protein utilizing the    principle of protein-dye binding, Anal. Biochem. 72, 248-254.-   Li, H., Oppert, B., Higgins, R. A., Huang, F., Zhu, K. Y.,    Buschman, L. L., 2004a. Comparative analysis of proteinase    activities of Bacillus thuringiensis-resistant and -susceptible    Ostrinia nubilalis (Lepidoptera: Crambidae). Insect Biochem. Mol.    Biol. 34, 753-762.-   Li, H., Oppert, B., Gonzalez-Cabrera, J., Ferré, J., Higgins, R. A.,    Buschman, L. L. and Zhu, K. Y. and Huang, F. 2004b. Binding analysis    of Cry1Ab and Cry1Ac with membrane vesicles from Bacillus    thuringiensis-resistant and -susceptible Ostrinia nubilalis    (Lepidoptera: Crambidae). Biochem. Biophys. Res. Commun. 323, 52-57.-   Schneider, J. C. Jenings A F, Mun D M, McGovern P M, Chew L C. 2005.    Auxotrophic markers pyrF and proC can replace antibiotic markers on    protein production plasmids in high-cell-density Pseudomonas    fluorescens fermentation. Biotechnology Progress 21, 343-348.-   Wolfersberger, M. G., Luthy, P., Maurer, A., Parenti, P., Sacchi,    F., Giordana, B., Hanozet, G. M., 1987. Preparation and partial    characterization of amino acid transporting brush border membrane    vesicles from the larval midgut of the cabbage butterfly (Pieris    brassicae). Comp. Biochem. Physiol. 86A, 301-308.-   US Patent Application No. 20080193974. 2008. BACTERIAL LEADER    SEQUENCES FOR INCREASED EXPRESSION-   US Patent Application No. 20060008877, 2006. Expression systems with    sec-system secretion.-   US Patent Application No. 20080058262, 2008. rPA optimization.

We claim:
 1. A transgenic corn plant that produces a Cry3Aa insecticidal protein and a Cry6Aa insecticidal protein, wherein said Cry3Aa protein consists of SEQ ID NO:2, and said Cry6Aa protein is full-length and consists of SEQ ID NO:3, wherein the combination of the Cry3Aa insecticidal protein and the Cry6Aa insecticidal protein is insecticidally effective against corn rootworm, and wherein the Cry3Aa insecticidal protein and the Cry6Aa insecticidal protein do not share a receptor binding site in corn rootworm gut.
 2. The transgenic plant of claim 1, wherein said plant further produces a third insecticidal protein selected from the group consisting of Cry3Ba, Cry34Ab, and Cry35Ab.
 3. A seed of the transgenic plant according to claim 1, wherein said seed comprises DNA encoding said proteins.
 4. A plurality of plants comprising a plurality of transgenic plants according to claim
 1. 5. The plurality of plants of claim 4, said plurality of plants further comprising non-Bt refuge plants, wherein said refuge plants comprise less than 40% of all crop plants in said plurality of plants.
 6. The plurality of plants of claim 5, wherein said refuge plants comprise less than 30% of all crop plants in said plurality of plants.
 7. The plurality of plants of claim 5, wherein said refuge plants comprise less than 20% of all crop plants in said plurality of plants.
 8. The plurality of plants of claim 5, wherein said refuge plants comprise less than 10% of all crop plants in said plurality of plants.
 9. The plurality of plants of claim 5, wherein said refuge plants comprise less than 5% of all crop plants in said plurality of plants.
 10. The plurality of plants of claim 4, wherein said plurality of plants lacks refuge plants.
 11. The plurality of plants of claim 5, wherein said refuge plants are in blocks or strips.
 12. A mixture of seeds comprising refuge seeds from non-Bt refuge plants, and a plurality of seeds of claim 3, wherein said refuge seeds comprise less than 40% of all the seeds in the mixture.
 13. The mixture of seeds of claim 12, wherein said refuge seeds comprise less than 30% of all the seeds in the mixture.
 14. The mixture of seeds of claim 12, wherein said refuge seeds comprise less than 20% of all the seeds in the mixture.
 15. The mixture of seeds of claim 12, wherein said refuge seeds comprise less than 10% of all the seeds in the mixture.
 16. The mixture of seeds of claim 12, wherein said refuge seeds comprise less than 5% of all the seeds in the mixture.
 17. A method of delaying or preventing the development of resistance to a Cry protein by a corn rootworm insect, said method comprising planting seeds to produce the plurality of plants of claim 4, wherein the plurality of plants expresses the Cry3Aa insecticidal protein and the Cry6Aa insecticidal protein.
 18. The plurality of plants of claim 4, wherein said plants occupy more than 10 acres.
 19. A plant cell of the plant of claim 1, wherein said plant cell produces said Cry3Aa insecticidal protein and said Cry6Aa insecticidal protein.
 20. A method of producing the plant cell of claim 19, said method comprising transforming the plant with a DNA encoding said Cry3Aa insecticidal protein and a DNA encoding said Cry6Aa insecticidal protein.
 21. A method of controlling a corn rootworm insect, said method comprising contacting said insect with the transgenic plant of claim 1, wherein the plant expresses an insecticidally effective amount of said Cry3Aa insecticidal protein and said Cry6Aa insecticidal protein. 